New spectroscopic methods are needed to determine the structural changes that occur in biological systems on the femtosecond (10-15 s) to nanosecond (10-9 s) time-scale. Photodamage to nucleic acids by ultraviolet (UV) light occurs in 100's of femtoseconds and numerous functions of peptides, proteins and oligonucleotides depend on structural fluctuations occurring over picoseconds to nanoseconds and longer. In each of these cases, there are few experimental techniques that can resolve the dynamic molecular structure of the evolving system. Our work aims to correct this by developing ultrafast Raman spectroscopy capable of collecting vibrational spectra, which can be directly related to molecular structure, of photochemically and thermally activated dynamics in biomolecules. Specific Aim #1 of this proposal will develop new femtosecond Raman instrumentation that can collect high-resolution vibrational spectra with time resolution better than 100 fs. This methodology will be applied to gain new understanding of the ultrafast dimerization of pyrimidines in DNA following excitation by UV light, as well as new fundamental understanding of the quantum mechanical nature of the excitation in nucleic acid polymers. With Specific Aim #2, we will develop new methodologies to impulsively initiate thermal unfolding of biopolymers. This will allow the numerous tools of time-resolved spectroscopy, which currently have applications limited to photochemistry, to unravel the dynamics of thermally driven secondary structural changes on the picosecond to nanosecond time-scale. Raman spectroscopy is uniquely positioned to contribute to these areas because of its ability to collect vibrational spectra of biopolymers over a wide spectral window without interference from the aqueous environment. Raman spectra of biopolymers exhibit particular peaks that are characteristic of the secondary structure of the polymer and the particular environment of the side chains or nucleic acids. Hence by collecting time-resolved Raman spectra as biochemical processes proceed, we can determine both the time-scales of formation and the structures of kinetic intermediates. This experimental work will complement the many theoretical predictions that have been made about ultrafast structural changes in photoexcited DNA and longer time-scale structural fluctuations. The proposed research is significant because of the breadth of photobiology that can be addressed by these techniques and the need for experimental probes of rapid structural changes important to biology and human health. PUBLIC HEALTH RELEVANCE (provided by applicant): The proposed research directly supports the mission of the NIH by helping to establish new understanding of the mechanisms of ultraviolet light damage to DNA, and its implication for public health. Ultraviolet light is one of the most prevalent environmental mutagens on earth, with significant deleterious effects on human health. These studies will also increase the capability of biomedical researchers to investigate the rapid structural motions important to the functions of proteins and DNA, thereby accelerating the development of new therapeutics.